The invention relates to an apparatus and its method of use for determining the presence or concentration of urea in a liquid sample, for example in laboratory autoanalyzers and preferably in single-use disposable cartridges adapted for conducting diverse real-time or near real-time assays of analytes.
In specific embodiments, the invention relates to the determination of urea in biological samples such as blood, blood components and urine.
Micro-fabrication techniques (e.g. photolithography and plasma deposition) are attractive for construction of multilayered sensor structures in confined spaces. Methods for microfabrication of BUN (Blood Urea Nitrogen) sensors, for example on silicon substrates, are disclosed in U.S. Pat. No. 5,200,051 to Cozzette et al., which is hereby incorporated by reference in its entirety. The sensor comprises a silicon chip with a silver/silver chloride electrode over which is a plasticized polyvinylchloride layer containing the ammonium ionophore nonactin. Over this layer is a layer of a film-forming latex material containing urease. Alternative ammonium ionophores include gramicidin D (Nikolelis and Siontorou; Ammonium ion minisensors form self-assembled bilayer, Anal. Chem. 68, 1735, 1996) and bicyclic peptides (Nowak; Design, synthesis and evaluation of bicyclic peptides as ammonium ionophores; Thesis, Worcester Polytechnic Institute, 2003).
The Cozzette et al., device operates in the standard potentiometric manner, where the enzyme urease converts urea from the sample to ammonium ions, these ions are detected by the ammonium-selective membrane covering the electrode. The electrical potential at the electrode is a logarithmic function of the ammonium concentration and thus the bulk urea concentration. By calibrating the sensor with known standards containing urea, the urea concentration in the sample can be estimated.
The enzyme carbonic anhydrase (CA) has been used in a carbon dioxide (pCO2) sensor, where it was added to the electrolyte layer to accelerate the CO2/H2CO3 aqueous equilibrium. This use of CA is well known and unrelated to urea sensing, see: Lindskog, S., Henderson, L., Kannan, K., Liljas, A., Nyman, P., and Strandberg, B.: Carbonic Anhydrase, The Enzymes 5, 587, 1971.
Botre, C. and Botre, F., (“Carbonic Anhydrase and Urease: An Investigation In Vitro on the Possibility of Synergic Action,”) Biochimica et Biophysica Acta, 997, 111-4, (1989), contains support for there being a physiological linkage between in vivo levels of urea and the production of CA enzyme activity. Botre teaches that placing an ammonia gas-sensing electrode, a carbon dioxide gas-sensing electrode, and a pH sensor into a solution containing either urease, or urease and carbonic anhydrase. It also teaches the addition of urea and a CA inhibitor (acetazolamide) to the solutions. These experiments are consistent with predictions based on the law of mass action, which are that the presence of carbonic anhydrase can influence (increase) the rate of hydrolysis of urea by urease, since CA effectively removes carbon dioxide from the system by moving it into the gas phase.
Specifically, the bicarbonate formed by the urease reaction is converted to carbon dioxide by CA, which then diffuses out of the liquid phase and into the air. This process has the effect of reducing the back reaction in which ammonium ions plus bicarbonate are converted to urea. Thus the net effect of the presence of CA in this system is to increase the rate of ammonium ion production.
Regarding the law of mass action, it is well known in the art of reversible enzymatic reactions, generically A=B+C, to add a reagent D which reacts with C, for the purpose of driving the reversible reaction in the direction of B (A. W. Adamson, A Textbook of Physical Chemistry, Academic Press (New York) 1973, chapter 7). While the present invention also seeks to use carbonic anhydrase to influence the reactivity of urease (UR), Botre does not suggest the present invention for at least the following reasons:
Botre is silent on analytical determination of the concentration of urea in a sample, and silent on analytical determination of urea in biological samples of clinical interest, e.g. a blood sample. Botre is also silent on the immobilization of UR and CA, as well as on potentiometric electrodes with immobilized enzymes.
Botre is silent on microfabrication of potentiometric electrodes for determining any analyte including urea, and on the use of the enzymes UR and CA in a system which does not permit the exchange of carbon dioxide from solution to an air space, as in a cartridge of the U.S. Pat. No. 5,096,669 incorporated herein their entirety by reference and other analytical systems where urea is measured electrochemically.
The concept of differential electrochemical e.g. potentiometric and amperometric, measurement is well known in the electrochemical art, see for example Cozzette, U.S. Pat. No. 5,112,455 and Cozzette, U.S. Pat. No. 5,063,081, both incorporated herein by reference in their entirety.
U.S. Pat. No. 5,081,063 discloses the use of permselective layers for electrochemical sensors and the use of film-forming latexes for immobilization of bioactive molecules, incorporated herein by reference. The use of poly(vinyl alcohol) (PVA) in sensor manufacture is described in U.S. Pat. No. 6,030,827 incorporated by reference. U.S. Pat. Nos. 6,030,827 and 6,379,883 teach methods for patterning poly(vinylalcohol) layers and are incorporated by reference in their entirety.
Gel electrophoresis of a typical commercial urease preparation, as shown in FIG. 14, indicates that prior art BUN sensors would not have included Carbonic Anhydrase as a significant impurity.